It is estimated that each year greater than 7 million people develop chronic, non-healing wounds in the United States. A majority of chronic wounds fall into three categories: pressure, venous and diabetic ulcers. The incidence is 0.78% of the population and the prevalence ranges from 0.18 to 0.32%. As the population ages, the number of chronic wounds is expected to rise (Crovetti, et al., Transfus. Apher. Sci., 30:145-51 (2004); Moreo, Case Manager, 16:62-3, 67 (2005); Supp, et al., Clin. Dermatol., 23:403-12 (2005); Mutoe, Am. J. Surg., 187:65S-70S (2004)). The National Pressure Ulcer Advisory Panel reports wide ranges of prevalence among patients in the United States. World-wide, in 2005, diabetes affected approximately 171 million people, including 20.8 million Americans, (7% of the population) according to 2005 NIH data. By 2030, these numbers are projected to double (Wild, et al., Diabetes Care, 27:1047-53 (2004)). Chronic wounds precede 84% of all diabetes-related lower-leg amputations (Reiber, et al., Diabetes Care, 22:157-62 (1999)). Therefore, understanding the pathogenesis and the ability to accelerate healing of these ulcers would have a major public health impact.
Skin integrity and its normal function depend on the ability of keratinocytes to maintain the barrier. In healthy epidermis, keratinocytes slowly proliferate in the basal layer and differentiate in the suprabasal layers. Basal keratinocytes are mitotically active. Once they leave the basal cell compartment they change their phenotype to differentiating. During the process of differentiation, they stop dividing, change the keratin production from K5/K14 to K1/K10, and begin to produce a number of insoluble proteins. At the end of the process they lose their nuclei and cross link their proteins giving rise to a cornified layer, forming a barrier. However, keratinocytes must respond very quickly to injury. In the case of injury, keratinocytes must inform each other that the bather has been broken and must be repaired (Freedberg, et al., J. Invest. Dermatol., 116:633-40 (2001); Tomic-Canic, et al., The epidermis in wound healing, Eds. Rovee and Maibach, CRC Press LLC, pps. 25-7 (2004); Morasses and Tomic-Canic, Biol. Cell, 97:173-83 (2005); Tomic-Canic, Wounds, 17:s3-6 (2005)). In response, they change their phenotype to activated (wound healing), alert the host defense mechanisms that the barrier has been broken and that pathogens may be intruding. As a response to their own signals, keratinocytes start migrating and proliferating. Epithelialization is an important component of wound healing, often used as its defining parameter (Brem, et al., Surgical Technology International, 161-7 (2003)). This process is governed by extra-cellular signals such as pro-inflammatory cytokines and growth factors (Freedberg, et al., J. Invest. Dermatol., 116:633-40 (2001); Tomic-Canic, et al., J. Dermatol. Sci., 17:167-81 (1998); Kupper, J. Invest. Dermatol.,  94:146S-150S (1990); Parks, Wound Repair Regen., 7:423-32 (1999); Mast, et al., Wound Repair Regen., 4:411-20 (1996)). To close the gap, keratinocytes must first “let go of their anchor”, i.e., loosen their adhesion to each other and to the basal lamina, and they must obtain the flexibility and ability to “grasp, hold and crawl” over the matrix freshly deposited by dermal fibroblasts. This requires rearrangement of the integrin receptors, reassembly of the associated actin cytoskeleton and the keratin filament network. Once the wound surface is covered by a keratinocyte monolayer, the proliferation signals cease and a new stratification process begins again.
Epidermal morphology of chronic ulcers differs from normal epithelial tissue and suggests that keratinocytes do not successfully complete either of the two possible pathways: activation or differentiation (Stojadinovic, et al., Am. J. Pathol., 167:59-69 (2005)). Instead, keratinocytes are caught in a ‘loop’ of trying, but not succeeding, to achieve either of the two processes. Non-healing keratinocytes of the chronic wound are marked by activation of glucocorticoid receptor (GR), induction of c-myc and nuclear presence of β-catenin, and de-regulation of EGF leading to increased proliferation and inhibition of migration (Brem, et al., Mol. Med, 13:30-9 (2007); Stojadinovic, et al., Am. J. Pathol., 167:59-69 (2005); Vukelic, et al., Wound Healing Society Meeting; Tampa, Wound Repair and Regen., A34 (2007)).
Glucocorticoids (GCs) act through glucocorticoid receptor(s) (GR) that may be active in all three cellular compartments: nuclear, cytoplasmic and membranous (Lee and Tomic-Canic, Molecular Mechanisms of Action of Steroid Hormone Receptors, Ed. Krstic, Research Signpost, pps. 1-25 (2002); Yudt, et al., Mol. Endocrinol., 15:1093-1103 (2001); Watson, et al., EMBO Rep., 6:116-9 (2005)). In addition to operating as a transcription factor directly binding promoter elements (genomic effect), GCs also interact with and affect the activity of a variety of transcription factors, thus affecting transcriptional potency of many signaling pathways, such as TNFα, IFN, EGF, etc (non-genomic effects) (Zhou, et al., Steroids, 70:407-17 (2005)). Therefore, the complexity of GCs action resides in multiple signaling routes that not always require transcriptional regulation. The genomic mechanism consists of several important interactions. GR binds to specific sequences in targeted promoters (response elements, GRE) (So, et al., PLoS Genet., 3:e94 (2007); Schoneveld, et al., Biochim. Biophys. Acta, 1680:114-28 (2004); Kumar and Thompson, J. Steroid Biochem. Mol. Biol., 94:383-94 (2005)). These sequences may mediate either activation (positive, GRE) or repression (negative, nGRE). Further, GR interacts with other transcription factors (AP-1, NF-kB) (De Bosscher, et al., Mol. Endocrinol., 15:219-27 (2001); Herrlich, Oncogene, 20:2465-75 (2001); Okabe, et al., Nippon Rinsho, 63:1654-59 (2005); Smoak, et al., Mech. Ageing Dev., 125:697-706 (2004)) or co-activators (such as GRIP-1, SRC-1) that modify its transcriptional signal (Cho, et al., Biochemistry, 44:3547-61 (2005); Li, et al., Mol. Cell Biol., 23:3763-73 (2003); Li, et al., Mol. Endocrinol., 20:1025-34 (2006); Ding, et al., Mol. Endocrinol., 12:302-13 (1998)). Lastly, this DNA-GR-co-regulator complex further interacts with histone modifying enzymes (acetyl or methyl transferases) that participate in chromatin remodeling and either activate or repress transcription (Li, et al., Mol. Cell Biol., 23:3763-73 (2003); Trotter, et al., Mol. Cell. Endocrinol., 265-266:162-7 (2007); Kagoshima, et al., Biochem. Soc. Trans., 31:60-5 (2003); Schurter, et al., Biochemistry, 40:5747-56 (2001); Koh, et al., J. Biol. Chem., 277:26031-5 (2002)).
In recent years, new aspects of GCs action have emerged. It was discovered that in addition to the effect that hormone bound GR has on transcriptional regulation, the receptor is capable of more rapid effects (non-genomic) such as changing the phosphorylation levels of other signaling molecules. The non-genomic effects are hormone dependant and mediated by the GR. Because they may affect signaling molecules, they may lead to transcriptional changes. Unlike in genomic effects, these transcriptional changes do not require direct interaction of GR with a promoter. For example, GCs can rapidly change the phosphorylation status of Lck/Fyn, interfere with PI3K and Akt, inhibit activity of LEF/TCF and may activate JNK, p38 and EGF signaling molecules (Croxtall, et al., Br. J. Pharmacol.,  130:289-98 (2000); Lowenberg, et al., Blood, 106:1703-10 (2005); Qi, et al., J. Neurosci. Res., 80:510-7 (2005); Smith and Frankel, J. Biol. Chem.,  280:2388-94 (2005); Leis, et al., Mol. Endocrinol., 18:303-11 (2004)).
GCs are major therapeutic agents that significantly inhibit epithelialization and wound healing, affecting millions of surgical patients as they are utilized for the treatment of inflammatory bowel disease and organ transplant as well as in the treatment of multiple skin diseases (Baumann and Kerdel, Fitzpatrick's Dermatology in General Medicine Vol. II, Eds. Irwin, et al., McGraw-Hill, pps. 2713-7 (1999); Trieu, et al., Mayo. Clin. Proc.,  80:1578-82 (2005); Kesisoglou and Zimmermann, Exp. Opin. Drug Del.,  2:451-63 (2005); Lemann, et al., Rev. Prat., 55:984-92 (2005); Schumacher and Chen, Am. J Med., 118:1208-14 (2005)). GCs block inflammation, repress immune system activation, act as growth-inhibitory agents and inhibit wound healing (Ehrlich and Hunt, Ann. Surg., 167:324-28 (1968); Beer, et al., Vitam. Horm., 59:217-39 (2000); De Bosscher, et al., J. Neuroimmunol., 109:16-22 (2000); Reed and Clark, J. Am. Acad. Dermatol.,  13:919-41 (1985)). Analyses of biopsies from patients suffering from chronic wounds revealed constitutive activation of GCs pathway and cortisol synthesis, suggesting that GCs play a role in the pathogenesis of chronic ulcers.
Most of the known GC effects are thought to be dermal (Schacke, et al., Pharmacol. Ther., 96:23-43 (2002)), however, much less is known about the effects of GCs on epidermis. GCs affect epidermal biology in many different ways, including cell-cell interaction, ECM molecules, and as immunosuppressive agents (Zettl, et al., Proc. Natl. Acad. Sci. USA, 89:9069-73 (1992); Guller, et al., Ann. NY Acad. Sci., 734:132-42 (1994); Cronstein, et al., Trans. Assoc. Am. Physicians, 105:25-35 (1992); Scheinman, et al., Science, 270:283-6 (1995)).
The molecular mechanisms involved in GCs-mediated inhibition of epithelialization are not well understood. A more complete understanding of the signaling pathways involved in GC-mediated inhibition of epithelialization would make it possible to design effective strategies to promote epithelialization and healing of chronic wounds.
Therefore, it is an object of the invention to provide compositions and methods to promote epithelialization and healing of chronic, non-healing wounds.
It is another object of the invention to provide compositions and methods to promote keratinocyte proliferation and migration at the leading edge of chronic, non-healing wounds.
It is yet another object of the invention to provide compositions and methods to inhibit or reduce induction of c-myc and nuclear presence of β-catenin in keratinocytes at the leading edge of chronic, non-healing wounds.